1. Field of Invention
The present invention relates to a nucleic acid construct comprising a pyripyropene biosynthetic gene cluster and a marker gene.
2. Background Art
It has been thus far proven that there are 18 types of naturally-occurring analogs from pyripyropene A to pyripyropene R in pyripyropenes, which analogs differ in structures of their side chains (Non-patent Document 1).
It has been disclosed that pyripyropenes have an ACAT inhibitory activity (Patent Document 1). Application thereof to treatment of diseases caused by cholesterol accumulation or the like is expected. Also, it has been disclosed that pyripyropenes have an insecticidal activity against Helicoverpa armigera larva (Non-patent Document 2), Diamondback moth larva (Patent Document 2), Tenebrio molitor (Patent Document 2) or aphids (Patent Document 3) and application thereof to insecticides is expected.
It has been known that pyripyropenes are produced as secondary metabolites by filamentous fungus. For instance, it has been disclosed that Penicillium coprobium PF1169 strain (Patent Document 4), Aspergillus fumigatus IFO-1289 strain (Patent Document 5), Eupenicillium reticulosporum NRRL-3446 strain (Non-patent Document 2) or Penicillium griseofulvum F1959 strain (Patent Document 2) each produces pyripyropenes.
Industrial production of pyripyropenes is carried out by culturing the above-mentioned production bacteria and collecting pyripyropenes. In general, the amount of secondary metabolism products produced by a separated naturally-occurring microorganism is small. In order to use this industrially, productivity of these desired products needs to be improved.
To improve the productivity of the desired products, studies for a method for culturing the desired product-producing microorganisms, studies for components of culture media and modifications of fermentation conditions such as addition of precursors, as well as modifications of bacterial strains using mutation by irradiation with ultraviolet light or mutagens have been carried out. Further, in addition to these methods, the improvement of the productivity using gene recombination has recently been carried out.
A general method in the improvement of the productivity by gene recombination is to enhance expression of a biosynthetic gene. For instance, by this method, a method for improving productivity of PF1022 substance produced by Agonomycetales is disclosed (Patent Document 6). In order to apply this method, it is required that the biosynthetic gene of a desired product be isolated and a method for transformation be established in a desired product-producing microorganism.
As for pyripyropenes, there are thus far no reports on isolation of their biosynthetic gene cluster. In addition, a method for transformation of a pyripyropene-producing fungus as a host has not been established. Therefore, it has thus far been difficult to introduce the biosynthetic gene cluster of pyripyropenes into the pyripyropene-producing microorganism and the improvement of the productivity by gene recombination is not able to be attained.